When will my service request be processed?
The Transgenic Core prioritizes all requests for service on a
"first-come,
first-serve" basis. This standard is applied to everyone equally, even
our faculty directors. The time between transgene
DNA
submission and microinjection is updated regularly. Typically ES
cell
work is scheduled one to three months ahead. Since the inception of the
Transgenic Core our policy has been that no project, whether it be gene
targeting in ES cells or a transgenic mouse model, will enter the work
queue until all of the required materials are provided. This includes
both
scientific materials, such as DNA samples and genotyping tests, and
paperwork,
such as approval to use animals in research, material transfer
agreements
and billing information.

How effective is the Transgenic Core?
Both transgenic and gene targeting efficiencies are excellent. We
guarantee
that at least three transgenic founders will be produced (the average
number
is 10). Since 1989 over 7000 transgenic founders were produced from
over
700 transgene constructs as of September 2003. These efficiency of
transgenic
production equals or exceeds values in the published transgenic
mouse
literature. The efficiency of all steps in gene targeting compare
favorably
with literature values. Multiple embryonic stem (ES) cell lines have
been
imported and screened for germline chimera formation. In addition, we
have
developed our own 129X1/SvJ ES cell line "Pat5". See the list
of ES cell lines. We have collaborated with investigators to
generate
70 new strains of mice (as of September 2003) from ES cells with
mutations
introduced by recombination with targeting vectors.

What about consultation?
We provide advice on all aspects of this technology from experimental
design to mouse breeding. We can provide protocols and training for
every
step in the process of generating transgenic or gene targeted mice. We
are ready to interact, our doors are open, please contact us with any
questions
Meet
the Staff.

What kind of paperwork is involved?
Any project that uses mice must be approved by the University
Committee on Use and Care of Animals. Concat the UCUCA for
innformation on how to apply for permision to use vertebrate animals in
research testing or instruction. The Unit for Laboratory Animal Medicine
provides animal housing and veterinary care. Investigators approved for
animal research are expected to provide ULAM with a shortcode that
can be used to pay for veterinary care and housing costs. Transgenic
Core submission forms require the following information:

What is a transgenic
mouse/rat?
A transgenic mouse or rat has a transgene in addition to its normal
complement of genes. A transgene is an artificial gene cloned in the
lab
by recombinant DNA technology and microinjected into fertilized mouse
or
rat eggs. Eggs are transferred into foster mothers for gestation.
Transgenic
progeny are bred to produce a line. Transgenes integrate randomly into
chromosomal DNA and are transmitted as a Mendelian trait.

What is involved in making transgenic mice/rats?
The Core is available for consultation on all phases of transgenic
research. The investigator designs and clones the transgene and
develops
a genotyping assay with single
gene copy sensitivity, usually PCR based. A genotyping assay that
detects
an endogenous single copy gene in the mouse
or
an endogenous gene in the rat is a
necessary
positive control. The assays for the transgene and the endogenous gene
are used to test all potential transgenic founder mice or rats. The
combination
of assays eliminates both false negatives and false positive
mis-identifications.
Transgene DNA is purified by the Transgenic
Core
for microinjection from a restriction digest supplied by the
investigator.
The Core microinjects DNA into fertilized (C57BL/6 X SJL)F2
eggs and transfers the eggs into pseudopregnant mice. Alternatively, we
will make transgenic mice in other genetic backgrounds, upon request.
We
have successfully made transgenic mice in C57BL/6 X SJL)F2,
FVB/N, C57BL/6, (C57BL/6 X DBA/2)F2, SWR, B10D2 congenic strains,
and mutant strains such as mnd2, Myo15sh2, and
C57BL/10ScSn-Dmdmdx/J.
When the pups are 2 weeks old, the Core applies ear tags and obtains
tail
biopsies from the mice. The Core will provide the tail
biopsies to the investigator. Investigators may prepare genomic
DNA from tail biopsies by hand or by any of the numerous kits on
the market (see Protocols). The
investigator will identify
the transgenic mice by PCR. The transgenic mice are transferred to
the investigator for breeding and analysis of transgene expression.

What can I do to maximize a successful transgenic
outcome?
We guarantee that you will receive 3 or more transgenic mice or rats,
however we can not guarantee transgene expression or transmission. The
best strategy is to use a promoter that is already well characterize in
transgenic mice or to employ very large flanking regions greater than
10
Kb. Alternatively, a bacterial artificial chromosome can be used to
direct gene expression. BACs are include over 100Kb of genomic DNA
sequence and often direct gene expression in a fashion which close
matches the expression of endogenous genes.

The yield of transgenics is optimized by injecting highly purified
linear
DNA fragments with overhanging ends. Remove as much vector sequence as
possible from the construct since prokaryotic sequences inhibit
transgene
expression. Although not a guarantee, demonstrated expression in a cell
line is a positive indicator of in vivo expression and provides
a rapid, inexpensive method to demonstrate that the transgene has been
constructed properly. Contact Thom
Saunders for more information on transgene design.

How many transgenic mice/rats will I get?
We guarantee that you will receive a minimum of three transgenic
founders,
often more. The purity of the microinjection DNA is the single most
important
factor which determines how many transgenic founders will be
produced.
Another very important parameter is the reliability and sensitivity of
the screen for the transgene.

What if my DNA construct is lethal?
If you believe that your construct may be lethal, we will co-inject
a neutral DNA fragment as a marker. If your transgene is not lethal
then
mice with both the transgene and neutral marker will be detected.
However,
if only mice with the neutral marker are detected then the construct is
likely to be embryonic lethal. Alternatively you choose to examine
transgenic
founders. Transgenic embryos can be analyzed at various developmental
ages
to determine the time of death during gestation.

What is the significant of transgene copy number?
For the majority of transgenes the copy number does not correlate with
expression level. The exceptions occur when large genomic fragments are
used to make transgenics. Examples include P1 clones (90 Kb), BACs (180
Kb,) or YACs (400 Kb). Some reports suggest that if you use locus
control
regions or matrix attachment regions around your transgene that you may
be able to insulate if form integration effects. Previous work in the
literature
has shown that attempts to produce low copy numbers by microinjecting
dilute
DNA does not affect copy number, but does reduce the overall yield of
transgenic
mice.

What if my transgene is too big?
The size of your transgene should not interfere with transgenic mouse
production. Large transgenes may be difficult to clone. You may wish to
consider BAC recombineering to produce large transgenes under the
control
of regulatory elements in the BAC. The Core has produced transgenics
from
Bacterial Artificial Chromosomes up to 180 Kb in size. There are
reports
in the literature of transgenic mice produced from the microinjection
of
90 Kb P1 clones, 248 Kb yeast artificial chromosomes, and even
microdissected
chromosome fragments.

What is a gene targeted mouse?
Gene targeted mice are derived from embryonic stem (ES) cells.
ES cells are manipulated in culture by introducing a targeting vector
that
is cloned in the lab by recombinant DNA technology. The targeting
vector
DNA precisely replaces a segment of chromosomal DNA (hence the name
"gene
targeting") in the ES cell. ES cells are injected into a normal mouse
blastocyst
where they mingle with the embryo' s cells to form the developing
mouse.
Up to 100% of the resulting mouse chimera can be formed from cells
descended
from the ES cells. ES cell-derived-mouse chimeras are bred to normal
mice
to produce progeny carrying the targeted gene which is transmitted as a
Mendelian trait.

What is involved making gene targeted mice?
The major stumbling block in this process is the identification of
ES cell clones that have undergone homologous recombination between the
targeting vector and the chromosome in the ES cell. To maximize a
successful
outcome: 1) make sure that the genomic DNA in your targeting
vector
is isogenic with the ES cell line question and 2) develop a
hybridization
screen that will detect your wild type gene in 2 ug of genomic ES cell
DNA. We encourage you to contact Thom
Saunders for a consult on your targeting vector design and on all
phases
of gene targeting research. The first step in the process is to obtain
and map and sequence a 129X1/Sv genomic clone of the gene of interest.
A 129X1/Sv library is available for screening from the core. The
investigator
designs and clones the targeting vector and purifies it for
electroporation
into embryonic stem (ES) cells. The Core has numerous plasmids designed
for targeting vector construction. The
targeting
vector is then electroporated into ES cells by Core personnel, clones
are
picked after drug selection, the clones are cryopreserved at -80 C
while
the investigator screens DNA from the clones to identify those which
have
undergone homologous recombination. Alternatively, the Core will train
you in ES cell culture and provide you with quality tested reagents and
space in the multi-user Mouse Embryonic Stem Cell Laboratory so that
you
can do the work yourself. After the investigator isolates euploid ES
clones
which have undergone homologous recombination with the targeting vector
they are microinjected into mouse blastocysts and transferred into
pseudopregnant
recipients. When the resulting pups are three weeks old, they are
scored
for ES cell contribution and transferred to the investigator for
breeding
and analysis.

What can I do to maximize a successful gene
targeting
outcome?

Characterize the genomic structure of your thoroughly.

Develop a sensitive screen for homologous recombination in the ES
cells.

Consider the biological consequence of the mutation you introduce.

We can guarantee that we will inject your embryonic stem (ES) cells
into
a minimum of 50 blastocysts for ES cell-mouse chimera formation.
Because
of the intrinsic variability in individual ES cell clones, we can not
guarantee
that chimeras will be produced or that they will transmit your targeted
gene through the germline. Therefore, we recommend that you provide at
least three clones for microinjection. In collaboration with other labs
on campus, we have successfully targeted over 60 genetic loci. We are
confident
in that we can work with you to genetically engineer new strains of
mice
that carry novel mutations of value to your research. The Core provides
plasmids for gene targeting, ES cell lines that have been tested for
germline
chimera formation, feeder cells for ES cell culture, FBS tested for ES
cell culture, and training in the exacting techniques required for
successful
ES cell culture. Contact Thom
Saunders
for
more information on gene targeting projects.

Transgenic and gene "knockout" animals that have been developed
using
NIH IRP (intramural research program) funds and resources will be
provided
to other laboratories following publication of descriptions of the
animals
in the peer reviewed literature. It is an obligation of NIH intramural
scientists to make such animals widely available for research purposes.
This can be achieved by making arrangements to send breeding pairs to a
central repository such as the Induced Mutant Resource at the
Jackson
Laboratory. This would assure the availability of clean,
genetically
characterized animals within a year's time. An attempt should be made
to
reduce duplication of effort by setting up collaborative experiments
whenever
possible; however, this should not be used as a mechanism to inhibit
the
distribution of animals.

These guidelines for the IRP are now in agreement with those of the
US Public Health Service (PHS) for the extramural community: "It is the
policy of PHS to make available to the public the results and
accomplishments
of the activities that it funds...Therefore, when these resources are
developed
with PHS funds and the associated research findings have been published
or after they have been provided to the agencies under contract, it is
important that they be made readily available for research purposes to
qualified individuals within the scientific community. This policy
applies
to grants, cooperative agreements, and contracts."